Pcdh cmv mcs ef1 gfp vector

To construct the lentivirus vector pCDH-HK2 expressing HK2, a full-length cDNA encoding the HK2 sequence was amplified from 293T cDNA and then cloned into the EcoR I/BamH I sites in the pCDH-CMV-MCS-EF1-GFP vector (System Biosciences, Mountain View, CA). Control lentiviral vector was used as a control. All plasmid sequences were confirmed by DNA sequencing. Lentivirus was generated by co-transfecting pCDH-CMV-MCS-EF1-GFP empty vector or pCDH-HK2 in 293T cells with Lipofectamine™2000 (Invitrogen). Target cells were infected overnight with empty vector or pCDH-HK2 in the presence of 8 μg/ml polybrene (Sigma-Aldrich). The following day, the cells were given fresh medium and allowed to grow for another 48 h. The transduction efficiency was measured by western blotting. The sorted cells were then characterized and used in further assays.

A full-length cDNA encoding the HK2 sequence which was amplified from 293T cDNA was cloned into the EcoR I/BamH I sites in the pCDH-CMV-MCSEF1-GFP vector (System Biosciences, Mountain View, CA, USA). Lipofectamine™2000 (Invitrogen, Carlsbad, CA, USA) was used for co-transfection of pCDH-CMVMCS-EF1-GFP empty vector or pCDH-HK2 in 293T cells to confirm the plasmid sequences. The LM3 and BEL-7402 cell lines were then infected overnight in the presence of 8 μg/mL polybrene (Sigma-Aldrich). The empty vector was regarded as the control. The transduction efficiency was measured by PCR and western blotting.